WO2006103406A1 - Methode d'obtention d'un ensemble de molecules cmh - Google Patents

Methode d'obtention d'un ensemble de molecules cmh Download PDF

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Publication number
WO2006103406A1
WO2006103406A1 PCT/GB2006/001110 GB2006001110W WO2006103406A1 WO 2006103406 A1 WO2006103406 A1 WO 2006103406A1 GB 2006001110 W GB2006001110 W GB 2006001110W WO 2006103406 A1 WO2006103406 A1 WO 2006103406A1
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WO
WIPO (PCT)
Prior art keywords
mhc
mhc molecules
peptide
subset
steps
Prior art date
Application number
PCT/GB2006/001110
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English (en)
Inventor
Nikolai Franz Gregor Schwabe
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Proimmune Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Proimmune Limited filed Critical Proimmune Limited
Priority to EP06726519.9A priority Critical patent/EP1879918B1/fr
Priority to US11/887,568 priority patent/US20090312528A1/en
Priority to DK06726519.9T priority patent/DK1879918T3/da
Publication of WO2006103406A1 publication Critical patent/WO2006103406A1/fr
Priority to US13/324,470 priority patent/US20120083591A1/en
Priority to US14/180,701 priority patent/US20140162907A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • the present invention relates to a method of producing a set of MHC molecules, and more precisely to a method of producing a set of MHC molecules which differ in their peptide bound in the binding groove.
  • the method allows for parallel and expedite provision of differing MHC molecules at high yields for each molecule.
  • MHC Major Histocompatibility Complex
  • MHC-peptide molecules are useful for detecting, separating and manipulating T cells according to the specific peptide antigens these T cells recognize. It has also been understood that the interaction between MHC molecules and TCRs across cell surfaces is multimeric in nature and that the affinity of a single MHC molecule for a given TCR is generally quite low. As a consequence, there has been an effort to develop multimeric forms of isolated or recombinant MHC-peptide molecules that have an increased functional avidity in order to make such molecules more useful in the applications described above. Various methods are known from the art to purify protein complexes and especially MHC molecules.
  • European Patent Application EP 812 331 discloses a multimeric binding complex for labelling, detecting and separating mammalian T cells according to their antigen receptor specificity, the complex having the formula ( ⁇ - ⁇ -P) n , wherein ( ⁇ - ⁇ -P) is an MHC peptide molecule, n is > 2, ⁇ comprises an ⁇ chain of a MHC I or MHC II class molecule, ⁇ comprises a ⁇ chain of an MHC protein and P is a substantially homogeneous peptide antigen.
  • the MHC peptide molecule is multimerised by biotinylating the C terminus of one of the ⁇ or ⁇ chain of the MHC molecule and coupling of MHC monomers to tetravalent streptavidin/ avidin.
  • This method requires the site directed enzymatic biotinylation of the MHC molecules near one of its carboxyl termini.
  • An enzyme recognition peptide sequence of around 14 amino acids is fused to the C-terminus of one MHC peptide chain proteins, which then allows for the complex to be biotinylated by using a biotinylating enzyme recognising this site.
  • MHC alpha and beta chain proteins are refolded in the presence of peptide and then purified by column chromatography. Biotinylation is achieved by incubating purified MHC peptide complexes in a suitable buffer in the presence of the biotinylating enzyme BirA.
  • concentration steps before and after the chromatography steps are required for purification and changing the purified MHC molecules in and out of the buffer required for biotinylation. These steps usually lead to considerable losses in protein, and are hence typically carried out in a scale as large as possible.
  • Standard production of monomeric biotinylated MHC peptide complexes as described by the NIAJD tetramer core facility as available on 31 March 2005 under the following URL: http ://www. verkes. emorv. edu/TETRAMER/pdf/Protocols .pdf involves concentration of typically 500ml of refold mixture on a stirred cell concentrator to approximately 7ml. This will be followed by buffer exchange into biotinylation buffer using a gravity driven desalting column. The MHC complexes are then biotinylated as described above. Purification is done thereafter first by size exclusion chromatography, which requires approximately 100 minutes run time. The recovered complexes are then concentrated in a centrifugal filtering device before loading onto an ion exchange chromatography column for final purification.
  • the present invention in its first aspect relates to a method of producing a set of MHC molecules, which set includes a plurality of subsets of MHC molecules.
  • the MHC molecules of each subset differ from MHC molecules of at least one, and preferably any other subset in at least one element selected from the group consisting of a peptide bound in the MHC binding groove, an MHC alpha chain protein and an MHC beta chain protein.
  • Said method comprises the steps of: a) providing the MHC molecules for each subset and loading said MHC molecules with said peptide by refolding in presence of said peptide or by peptide exchange, and b) purifying substantially in parallel the subsets of MHC molecules as obtained in step a) by a chromatographic method using a stepped gradient for elution.
  • step a) the product of step a) is subjected to step b) without prior concentration.
  • the invention in its second aspect also relates to a set of MHC molecules produced by the above method.
  • the method of the invention by avoiding concentration steps and by parallel processing of a plurality of subsets - allows for providing a set of MHC molecules at high yields and high diversity of the subsets. This makes available such sets of MHC molecules for screening applications at reasonable costs.
  • the prior art processes essentially allow for producing one purified MHC molecule at a time. They are also carried out in rather large scale so that a sufficient amount of material can be recovered after all concentration, buffer exchange and purification losses. Both the requirement of large scale and high losses were up to now uneconomical for providing MHC molecules to be used in applications which may e.g. require evaluating 10 or more MHC molecules and possibly several 10s or even 100s of such molecules of e.g. differing specificity (as determined by differing peptides being bound in the MHC binding groove).
  • the invention method is based on the idea that various MHC molecules, though differing slightly e.g. in the peptide, are still usually very similar in their overall physical properties such as their isoelectric point, molecular weight, conformation and hydrophobicity. This allows their purification by applying essentially the same or very similar methods and conditions. This circumstance creates a unique opportunity for purifying MHC molecules of many different specificities of the individual subsets of MHC molecules substantially in parallel in a simplified but effective purification procedure. Here, parallel processing reduces manufacturing and hence product costs.
  • substantially in parallel means that the individual subsets are subjected to the same or similar process steps in close timely relationship, and preferably at substantially the same time or simultaneously. This does not require all of the subsets being processed at the same time, but is intended to include scenarios, wherein e.g. a first group of subsets is processed first and a second group is processed thereafter, and so on.
  • in parallel means a simultaneous processing of all subsets, even more preferably under similar or identical conditions.
  • Parallel processing may e.g. be in the same device (such as a centrifuge or a vacuum manifold) or by using parallel-operated equipment.
  • Substantially parallel processing occurs essentially in step b), i.e. during purification.
  • at least one of the purification steps of loading, washing and elution is carried out in parallel.
  • at least elution/recovery is carried out in parallel. More preferably, at least washing and elution are parallel.
  • Providing the MHC molecules for each subset or loading of MHC molecules with peptide of step a) needs not be parallel for each subset, but can of course be in parallel, if technically feasible and/or desirable.
  • the type of chromatography will usually be liquid chromatography.
  • step b) typically involves the steps of
  • elution is by using a stepped gradient.
  • a stepped gradient for elution within the scope of the invention means changing the composition or property of the solution in which the chromatography matrix is immersed in one or more discrete steps as opposed to changing it continuously.
  • a continuous gradient may for example be created by analogue operation of a mixing valve or quasi-continuous change in mixing of two or more solvent components under digital computer control, such as in a computer operated liquid chromatography system.
  • a stepped gradient for elution will usually involve preparing separate aliquots of solutions with different properties (such as differing salt concentration) which may be applied in a step-wise sequence one after another until elution occurs.
  • the number of steps in which this gradient is applied will depend on the chromatographic method chosen, tolerable losses and the desired purity.
  • the stepped gradient will at least include one step and typically less than 50 steps, preferably less than 10 steps, more preferably less than 2 steps and most preferably only one step.
  • the MHC subsets differ from at least one, and preferably any other subset in at least one element selected from the group consisting of a peptide bound in the MHC binding groove, an MHC alpha chain protein and an MHC beta chain protein.
  • the differing subsets taken together comprise the set to be provided by the present invention.
  • Each subset may be processed in an individual batch. This limitation does not exclude the possibility of individual subsets being present in more than one batch, provided that the set includes at least two differing subsets.
  • the MHC molecules of the subsets differ with regard to their peptides and are loaded with a peptide specific for the respective subset in step a).
  • said peptides are substantially homogenous in each subset.
  • the set as produced may comprise at least 2, preferably at least 4 and more preferably at least 10 subsets of MHC molecules. It also preferably comprises up to 96, preferably up to 48 and more preferably up to 24 subsets of MHC molecules. The number of subsets may depend on the precise method of chromatography chosen and the device used for implementing the same.
  • the chromatographic method is one using an adsorption matrix selected from the group consisting of resins, beads, and membranes (such as a porous membrane adsorption chromatography matrix).
  • Column designs are preferred over batch designs (e.g. batch designs where the adsorption matrix is directly added to the receptacle containing the solution to be purified), due to ease of handling and availability of equipment.
  • Various types of binding properties may be exploited such that the method may be chosen from the group consisting of affinity chromatography, ion exchange chromatography, iso-electric focussing chromatography, hydrophobic interaction chromatography, hydroxyl-apatite chromatography, and reverse phase chromatography; ion exchange chromatography being most preferred.
  • the adsorption matrix is a porous membrane adsorption matrix, such as described in WOOl 19483. More preferably such matrix is an ion exchange chromatography matrix.
  • the ion exchange chromatography method used is preferably anion exchange chromatography and most preferably uses a basic ion exchanger such as a resin comprising quaternary ammonium groups (DEAE).
  • a basic ion exchanger such as a resin comprising quaternary ammonium groups (DEAE).
  • Commercial ion exchange resins suitable for use in the present invention are e.g. those sold under the trademarks Sepharose ® , MonoBeads ® from GE Healthcare (formerly Amersham Biosciences).
  • the purification step b) comprises the steps of bl) loading the product of steps a) on the adsorption matrix, e.g. the column, b2) washing the loaded adsorption matrix (e.g. the column) at least once, and b3) eluting the adsorption matrix (e.g. the column) by applying a stepped gradient, b4) recovering the desired subset of MHC molecules in the eluate of step b3), and b5) optionally repeating steps bl to b4, individually or in combination, until the desired purity of the product is obtained.
  • the method of the invention will yield a purified set of MHC molecules, wherein impurities are removed from the MHC molecules in each subset during the wash step(s) before elution and/or by remaining bound to the matrix after the elution step(s).
  • the nature of the impurities that typically remain on the column after the elution step(s) is usually larger protein aggregates.
  • the method of the invention also allows for efficient buffer exchange of the MHC molecules in each subset from the buffer solution that they are provided in in step a) to the buffer solution they are eluted in.
  • the method may serve as a simultaneous means of concentration of the MHC molecules in solution in each subset from the volume of solution that they are provided in in step a) to the volume of solution they are eluted in.
  • the stepped gradient is preferably selected from any suitable gradients depending on the chromatographic method chosen. It will typically be selected from the group consisting of a salt gradient, a pH gradient or a gradient employing a denaturing or chaotropic agent, and mixtures thereof. Most preferably the stepped gradient is a one-step gradient such as a one- step salt gradient.
  • concentrations of the gradient are likewise chosen depending on the chromatographic method chosen and adsorption strength.
  • concentrations of the gradient are likewise chosen depending on the chromatographic method chosen and adsorption strength.
  • a salt gradient of at least 100 mM salt, preferably 200 to 500 mM salt is used. Most preferably the salt is NaCl.
  • step a) is loaded on the adsorption matrix - in the following reference will be made to columns only, though it is to be understood that the same or comparable conditions will apply to other types of adsorption matrix as well - under any suitable conditions as to capacity, number of loading steps, buffer solution, flow rate, pH and temperature.
  • the column is then subjected to washing and elution.
  • the solution used for at least one of the steps of purification selected from loading, washing and elution is any suitable solution such as an aqueous solution of pH above 7, preferably above 7.5. Identical solutions are typically chosen for the wash buffer and the loading buffer, but need not be. Elution occurs by using the stepped gradient as above.
  • the entire sequence of purification steps in b) (bl to b4) may be repeated individually (e.g. more than one washing step applied) or in combinations (sequence bl to b4 is repeated), as desired.
  • the conditions for carrying out at least one of the steps of purification in b) are substantially identical for at least two subsets and more preferably for all subsets.
  • the MHC molecules of each subset are provided and loaded with the desired peptide of interest in step a) by refolding MHC alpha and beta chain proteins of said MHC molecules in the presence such peptide as known in the art, e.g. by following a protocol described in Garboci et al, PNAS 89 (1992), 3429-3433 or the NIAID tetramer core facility protocol (supra). hi both cases the MHC a and ⁇ chain proteins are expressed in a bacterial host and obtained from inclusion body material.
  • the MHC molecules of each subset are provided in step a) by exchanging the peptide bound in said MHC molecules under suitable conditions to load the desired peptide as known in the art, such as disclosed in WO9310220 and T.O. Cameron et al, J. Immunol. Meth. 268 (2002), 51-69.
  • the MHC molecules for each subset are obtained by expression in a eukaryotic host and they may contain no peptide, peptide endogenous to the host cell or other irrelevant peptide before loading of the desired peptide of interest for each subset.
  • providing the MHC molecules for each subset and loading with the said peptide of interest may take place simultaneously or in sequence.
  • the MHC molecules may be selected from MHC monomers and oligomers.
  • oligomer is intended to include any molecule comprising more than one MHC monomer, each monomer consisting of alpha chain protein, beta chain protein and peptide bound in the binding groove. Examples of such oligomers are dimers to decamers, especially dimers, tetramers, pentamers and decamers. Oligomeriza- tion may occur as disclosed in the above references or in WO9310220, GB2392158 all of which are incorporated by reference.
  • the MHC molecules are MHC monomers, which have been bioti- nylated, preferably before subjecting them to step b).
  • the MHC monomers have been biotinylated on either their alpha or beta chain protein even before loading of the peptide either by refolding or peptide exchange in step a).
  • Biotinylation of the MHC monomers can be achieved as known in the art, e.g. by attaching biotin to a specific attachment site which is the recognition site of a biotinylating enzyme. Reference is in this regard made e.g. to EP 812 331.
  • biotinylation is carried out on the desired protein chain in vivo as a post translational modification during protein expression of such protein chain in the expressing host cells as described in WO9504069.
  • the method of the invention further includes the step of oligomerizing the MHC monomers to yield the desired oligomer. Such step of oligomerization may occur after purification. Alternatively it may occur before purification, either before step b) and after step a), or alternatively before or during step a).
  • the method of the invention may further include a step of purifying the a and ⁇ chain proteins of the MHC molecules before providing the MHC molecules in step a).
  • Purification of the a and ⁇ chain proteins as such or aligned to an MHC molecule may be carried out by any conventional method and preferably by ion exchange or affinity chromatography.
  • the driving force for at least one of the purification steps selected from loading, washing and elution is typically selected as appropriate for the method and device of chromatography chosen. It is preferably selected from gravitational forces, a centrifugal force applied by centrifuging the chromatography column or a pressure drop caused through applying a vacuum to one end of the column.
  • the plurality of subsets is purified substantially simultaneously in the same centrifuge or on a vacuum manifold.
  • vacuum manifolds are known in the art and are commercially available e.g. from Qiagen under the trade name QIAvac 24 plus, which is a 24 place manifold.
  • a commercially available centrifuge is used for spinning of appropriate columns.
  • the chromatography column is mounted directly on the receptacle receiving the eluate during the elution step b4).
  • Corresponding spinning columns are commercially available e.g. from Vivas- cience under the trademark VivaPure ® such as the VivaPure ® Q Mini H column.
  • the receptacle may also be a microcentrifuge tube suitable for holding between 0.5 to 2 ml volumes of solution such as those known in the art as Eppendorf ® tubes.
  • the method of the invention is also characterised in that preferably no concentration steps are carried out before subjecting the product obtained from step a), i.e.
  • step b) the product of step a) is subjected to step b) essentially as is or after an oligomerization thereof.
  • step b) may further be subjected to one or more steps selected from a labelling reaction, lyophilization, immobilisation on a solid surface or incorporation into a lipid (bi)layer or quality control e.g. by SDS-PAGE, immune precipitation or other. Any of these steps may likewise be carried out in parallel for the subsets, but need not be so.
  • the method of the present invention is especially suited to be carried out in small scale.
  • small scale batches of 200 ⁇ l to 500 ml per subset, preferably 1 to 10 ml per subset are meant. These can be handled in the above mentioned equipment which itself is commercially available.
  • the MHC molecules are selected from the group consisting of MHC Class I, MHC Class ⁇ , homo-oligomers thereof, hetero oligomers thereof and mixtures of the same.
  • the MHC proteins maybe from any vertebrate species, e.g. primate species, particularly humans; rodents, including mice, rats, hamsters, and rabbits; equines, bovines, canines, felines; etc. Of particular interest are the human HLA proteins, and the murine H-2 proteins.
  • HLA proteins include the class II subunits HLA-DP ⁇ , HLA-DP ⁇ , HLA-DQ ⁇ , HLA- DQ ⁇ , HLA-DR ⁇ and HLA-DR ⁇ , and the class I proteins HLA-A, HLA-B, HLA-C, and ⁇ 2 -microglobulin.
  • HLA-DP ⁇ HLA-DP ⁇
  • HLA-DP ⁇ HLA-DP ⁇
  • HLA-DQ ⁇ HLA- DQ ⁇
  • HLA-DR ⁇ and HLA-DR ⁇ HLA-A
  • HLA-B HLA-DQ ⁇
  • HLA-DR ⁇ HLA-DR ⁇
  • HLA-A proteins HLA-A, HLA-B, HLA-C, and ⁇ 2 -microglobulin
  • murine H-2 subunits include the class I H-2K, H-2D, H-2L, and the class II I-A ⁇ , I-A ⁇ , I-E ⁇ and I-E ⁇ , and ⁇ 2-microglobulin.
  • non-classical MHC molecules such as CDl, HLA-E, HLA-F, HLA-G, QaI, and CDl.
  • the CDl monomer may instead of the peptide have a lipid such as a glycolipid bound in its groove.
  • the present invention is also applicable to the situation where a lipid instead of a peptide is bound and the skilled worker will be capable of translating the above protocols to this situation.
  • the MHC peptide chain proteins correspond to the soluble form of the normally membrane-bound protein.
  • the soluble form is derived from the native form by deletion of the transmembrane and cytoplasmic domains.
  • the soluble form will include the ⁇ l, ⁇ 2 and ⁇ 3 domains of the ⁇ chain protein and beta 2 microglobulin, respectively.
  • the soluble form will include the ⁇ l and ⁇ 2 and ⁇ l and ⁇ 2 domains of the ⁇ chain protein and ⁇ chain protein, respectively.
  • the MHC molecule may in one or more of the proteins chains or peptide comprised therein further comprise one or more additional domains such as one or more linkers, a tagging domain and a purification domain.
  • the additional domain(s) may e.g. be provided on the multivalent entity in case of oligomers or on the peptide, the MHC alpha and/or beta chain proteins, respectively.
  • the present invention in a second aspect thereof relates to a set of MHC molecules, obtained according to the method as described above.
  • the plurality of subsets in this case differ from each other by the peptide bound in the MHC peptide binding groove of each subset.
  • the subsets differ with regard to specificity of otherwise identical MHC molecules.
  • HLA-A*0201 with 24 different binding peptides are set up according to the protocol as described in Garboci et al. (supra), by scaling down the refold method described therein to the volume of interest.
  • Solubilised HLA-A*0201 alpha chain protein and beta 2 microglobulin were obtained as described in the reference with the modification that the alpha chain protein was obtained in a pre-biotinylated form following the protocol described in WO9504069 by fusing a biotinylation peptide to the C-terminal end of the alpha chain protein.
  • this protocol uses stirring of the refolding mixture, vortexing is used following preparation of the refolding mixture and after the addition of each of the protein chains and the binding peptide. Refolds are incubated overnight at 4°C.
  • Resin wash step 2 Addition of 400 ⁇ l loading buffer to column and centrifugation for 5 min as above.
  • Sample elution Addition of 400 ⁇ l elution buffer (2OmM Tris pH 8.0 + 30OmM NaCl) to column and centrifugation for 5 min as above.
  • Biotinylated MHC monomers are now ready for coupling to streptavidin in a variety of applications. In one application they can be coupled to R-PE labelled streptavidin in a 4:1 molar ratio of biotinylated monomer to streptavidin molecule to provide MHC tetramers that can be used to detect antigen specific T cells in flow-cytometry.

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Abstract

La présente invention concerne une méthode d'obtention d'un ensemble de molécules CMH, et plus particulièrement une méthode d'obtention d'un ensemble de molécules CMH qui diffèrent dans leur liaison peptidique dans le sillon de liaison. Cette méthode autorise une synthèse rapide et parallèle de molécules CMH différentes, avec de forts rendements pour chaque molécule.
PCT/GB2006/001110 2005-04-01 2006-03-28 Methode d'obtention d'un ensemble de molecules cmh WO2006103406A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP06726519.9A EP1879918B1 (fr) 2005-04-01 2006-03-28 Methode d'obtention d'un ensemble de molecules cmh
US11/887,568 US20090312528A1 (en) 2005-04-01 2006-03-28 Method of Producing a Set of MHC Molecules
DK06726519.9T DK1879918T3 (da) 2005-04-01 2006-03-28 Fremgangsmåde til fremstilling af en gruppe mhc-molekyler
US13/324,470 US20120083591A1 (en) 2005-04-01 2011-12-13 Method of producing a set of mhc molecules
US14/180,701 US20140162907A1 (en) 2005-04-01 2014-02-14 Method of producing a set of mhc molecules

Applications Claiming Priority (2)

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GB0506706.1 2005-04-01
GB0506706A GB2424645B (en) 2005-04-01 2005-04-01 Method of producing a set of MHC molecules

Related Child Applications (2)

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US11/887,568 A-371-Of-International US20090312528A1 (en) 2005-04-01 2006-03-28 Method of Producing a Set of MHC Molecules
US13/324,470 Continuation US20120083591A1 (en) 2005-04-01 2011-12-13 Method of producing a set of mhc molecules

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WO2006103406A1 true WO2006103406A1 (fr) 2006-10-05

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Cited By (2)

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EP2264067A1 (fr) * 2009-06-18 2010-12-22 Hla-G Technologies Multimères de HLA-G alpha 1 et leurs utilisations pharmaceutiques
WO2010146094A1 (fr) * 2009-06-18 2010-12-23 Hla-G Technologies Multimères alpha 1 de hla-g et leurs utilisations pharmaceutiques

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GB2442048B (en) * 2006-07-25 2009-09-30 Proimmune Ltd Biotinylated MHC complexes and their uses
GB2440529B (en) * 2006-08-03 2009-05-13 Proimmune Ltd MHC Oligomer, Components Therof, And Methods Of Making The Same
KR102655456B1 (ko) 2021-09-09 2024-04-05 백영완 전기설비 수배전함 내의 화재 영상 감지를 위한 지능형 영상 감지 시스템

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